Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

Figures

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  Fig 1. Ang-II induces ACE translocation to the nucleus. (A) Internalization of AT1 and ACE in the presence of 4 nM 3H-Ang-II. Data are shown as mean from three independent experiments, each performed in duplicate. (B) CHO-ACE and CHO-AT1 cells present the same relative protein level of each respective receptor. (Values are mean ± S.E.M, *p<0.05, n = 63 individual experiments). (C) Representative confocal images of internalized Ang-II-FITC (1 μM) in CHO-ACE and CHO-AT1 cells after 30 seconds of Ang-II stimulation. DAPI (blue) and Wheat Germ Agglutinin (red), scale bar = 10 μm. On the right, quantification of internalized Ang-II-FITC is presented. Values are mean ± S.E.M, *p<0.05, n = 6. (D) Representative confocal images of unstimulated CHO-ACE and CHO-AT1 cells, labeled for DAPI and WGA. (E) Immunolocalization of ACE after stimulation with Ang-II (1μM), for the indicated times. ACE is shown in green, actin filaments in red, and nucleus in blue (DAPI). Right panel represents a 3D reconstruction of CHO-ACE cell after 15 minutes of incubation with Ang-II (1μM). Scale bar = 10μm. (F) Western blotting of nuclear and non-nuclear protein fractions from CHO-ACE cells, before (control) and after…

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  Fig 2. Molecular interaction between ACE and PLC by computational analysis in silico. (A) Representation of the ten top ranked docking poses for ACE (blue) with PLCβ3 protein superimposed (the color of the PLC protein pose correspond to the colors of the labels). (B) Structure of the complex between ACE (blue) and PLC-β3 (Pose 9, red). The binding energy for the best docking pose of ACE and PLC-β3, Pose 9, is -194.06 kcal/mol. C) Amino acid residues located at the interface between the best docking pose of PLC-β3 (left panel) and ACE (right panel), Pose 9, explored through docking protocols.

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  Fig 3. Effect of Ang-II on intracellular Ca2+ signaling in CHO-ACE cells. (A) Western immunoblotting to confirm the silencing of PLC isoforms. Densitometric analysis are shown on (B) for PLCβ3 and (C) for PLCγ1. (D) Quantitative representation of intracellular [Ca2+] in CHO-ACE cells transfected with siRNA-PLCβ3 and PLCγ1 using Lipofectamine and stimulated with Ang-II (1μM). (E) Line scanning of Ca2+ signal in CHO-ACE. Ang-II promotes Ca2+ increase with greater intensity in the nuclear region. (F) Time course of Ca2+ signaling in the nucleus (red traces) and cytosol (blue traces). (G) Quantification of fluorescence intensity signal in the nucleus and cytosol. Values are mean ± S.E.M., (* p<0.01), 45 cells, n = 3 individual experiments.

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  Fig 4. Cell proliferation induced by Ang-II/ACE involves clathrin- mediated internalization process. (A) Cell growth assay of CHO-ACE cells 12, 24 and 48 hours after stimulation with Ang-II (1μM), triplicate in 3 individual experiments. (B) Western blot to confirm the silencing of clathrin (upper panel) and densitometry analysis (bottom panel). Mean ± S.E.M., n = 5 (* p<0.05). (C) BrDU incorporation is decreased in CHO-ACE cells transfected with siRNA-Cla (Clathrin) and stimulated with Ang-II (1μM). Mean ± S.E.M., n = 6 (* p<0.05).

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  Fig 5. Ang-II stimulates proliferation in melanoma cells. (A) Real-Time PCR analysis for expression of ACE in melan-a and TM-5 cells. (B) ACE activity measured by cleavage of Abz-FRK(Dnp)P-OH in melan-a and TM-5 cells. For A and B, mean ± S.E.M, n = 6 (**p<0.01). (C-D) BrDU uptake assay 24 hours after stimulation with Ang-II (1μM) in the presence of lisinopril (1μM), showing inhibition of cell proliferation in TM-5 cells (D) but not in melan-a cells (C). Mean ± S.E.M., n = 6. (*p<0.05).

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  Fig 6. ACE silencing inhibits the proliferative effect of Ang-II in melanoma cells. (A) Western bot (upper panel) to confirm the silencing of ACE and densitometry analysis (bottom panel). Mean ± S.E.M, n = 8. (***p<0.01 compared to respective columns). (B) BrDU uptake in Tm5 cells silenced for ACE and stimulated for 24 hours with Ang-II (1μM), showing a decrease in BrDU incorporation in the absence of ACE. Mean ± S.E.M., n = 12 (*p<0.05; ns = non-significant).

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  Fig 7. Ang-II promotes cellular migration and reduces focal adhesion formation in melanoma cells. (A-B) Wound healing assay using TM-5 cells stimulated with Ang-II (1μM). C) Representative confocal images of TM-5 cells double-labeled with vinculin (green) and phalloidin (red). Scale bar = 10 μm. (D-E) Quantification of the focal adhesion formation. Mean ± S.E.M., n = 6 (* p<0.05).